1. Field of the Invention
This invention relates to means for detecting an antigen or an antibody in a fluid sample based on passive hemagglutination. In one aspect, the present invention relates to the detection of chorionic gonadotropin in a urine specimen and therefore finds particular application in the detection of pregnancy.
Hemagglutination may be generally referred to as the clumping or aggregation of red blood cells. The clumping or aggregation of erthrocytic carrier particles which are incorporated with an immunochemically active substance in the presence of an appropriate immunochemical counterpart is referred to as passive hemagglutination. As used herein, the term immunochemically active substance refers to one member of the group consisting of an antigen or an antibody thereto and the term immunochemical counterpart refers to the other member.
There are basically two immunological test methods involving passive hemagglutination, direct passive hemagglutination tests and passive hemagglutination inhibition tests. Both methods involve the use of a passive hemagglutination indicator which comprises an erythrocytic carrier particle having an immunochemically active substance attached thereto. The indicator is fabricated such that passive hemagglutination will occur when an immunochemical counterpart to the carrier-incorporated immunochemically active substance is contacted with the indicator.
In direct passive hemagglutination tests, the presence of an immunochemically active substance in a sample may be determined by combining, in a vessel having a concave bottom, a quantity of sample to be tested and a passive hemagglutination indicator which comprises the immunochemical counterpart. If present, the immunochemically active substance reacts with the carrier-incorporated counterpart to form a smooth, substantially homogeneous, hemagglutination deposit. In the absence of the immunochemically active substance, the indicator settles to form a visible disc or ring on the bottom of the test vessel.
In passive hemagglutination inhibition tests, the presence of an immunochemically active substance in a sample may be determined by combining, in a vessel having a concave bottom, a predetermined quantity of the counterpart, and a passive hemagglutination indicator which comprises the immunologically active substance to be determined. Since the affinity of the counterpart for the immunochemically active substance free in solution is greater than its affinity for the immunochemically active substance incorporated with the carrier, hemagglutination will occur only when the substance to be determined is present in the sample in an amount less than that capable of reacting with all of the counterpart present. Thus, in contrast to the direct method, the presence of the substance to be determined, rather than its absence, is indicated by the appearance of a visible ring or disc comprised of settled indicator particles.
2. Description of the Prior Art
It is well known that conventional passive hemagglutination inhibition test systems are characterized as having an unacceptable level of false negative results. In reading the test results, the lack of appearance of a visible indicator sediment due to the occurrence of hemagglutination is recorded as a negative result. Theoretically, where there is a sufficient amount of the immunochemically active substance to be determined in the sample tested to react with the quantity of counterpart present, hemagglutination should not occur as there would be no free counterpart available to react with the hemagglutination indicator. However, it has been found that hemagglutination may occur even where there is no available free counterpart, that is, where there is a sufficient level of the substance to be determined in the sample to bind all the free counterpart. Such spontaneous or non-specific hemagglutination therefore yields a false negative result.
The problem of false negative results in passive hemagglutination inhibition test systems for detecting gonadotropins in urine has been previously recognized in British Pat. No. 979,759. It was suggested that the cause of spontaneous agglutination was an excess of calcium ions. The hypothesis was that the incidence of false negative results is a function of the age of the urine specimen tested, since the observation was made that calcium ion level increased with the age of the urine. The solution proposed in British Pat. No. 979,759 was to carry out the test reaction in the presence of a calcium chelating agent in order to bind all the calcium ions present in the test sample and thereby to prevent calcium ion-induced, non-specific agglutination. The preferred chelating agents proposed were ethylenediamine tetraacetic acid, citric acid and its alkali metal and ammonium salts.